FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Abstract

Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.

Synthesis and Biological Evaluation of RGD–Cryptophycin Conjugates for Targeted Drug Delivery

“…The RGD–cryptophycin conjugates consist of the highly cytotoxic payload, cryptophycin-55 glycinate, and c (RGDfK) peptide connected through the protease-cleavable Val-Cit dipeptide linker, which is extensively used in the ADC field [44]. This peptide-based linker displays an excellent balance between high stability in circulation and rapid intracellular cleavage in the presence of lysosomal proteases, mainly cathepsin B [45] and other cysteine cathepsins [46,47]. In order to achieve efficient release of the cytotoxin in an active form, two different self-immolative spacers were inserted between the drug and the cleavage site: the para -aminobenzyloxycarbonyl (PABC) spacer, which undergoes 1,6-elimination, or the Gly-Pro dipeptide unit, which was designed to decompose by diketopiperazine formation [48,49].…”

The Analysis of Key Factors Related to ADCs Structural Design

“…Although the linker may be not directly correlated with the final potency of ADC (Lee et al, 2018b), the potency of ADC is dictated by the concentration of payload accumulated in tumor cells, and the payload release is determined by the stability of the linker. Thus, the linker is crucial for a perfect ADC, and it determines the stability, efficacy, and even the ability to overcome MDR.…”

“…The non-cleavable linkers are not only more stable to escape from the off-target toxicities than cleavable linkers (Lu et al, 2016), but also may overcome the barrier of multiple-drug resistance (MDR) (Shefet-Carasso and Benhar, 2015; Beck et al, 2017; Nasiri et al, 2018) for the reason that the payload connected with polar amino cannot be a substrate of MDR1, which will improve the MDR phenomenon. However, the non-cleavable linkers need a more elaborate process to produce activity such as the internalization and metabolism of the antibody in the lysosome, which is a prerequisite to release active payloads to exert killing activity (Rosenberg, 2006; Lambert and Berkenblit, 2018), and the polar amino-linker-payload also needs a distinct transporter to carry it from the lysosome to cytoplasm to work (Hamblett et al, 2015; Kinneer et al, 2018; Lee et al, 2018b), which makes the design of ADC more complex to limit the utilization of non-cleavable linkers. The cleavable linkers are more vulnerable to lead to off-target toxicities, but the process of exerting effects is more comprehensible thus researchers are dedicated to modifying the cleavable linkers to overcome their weakness and to increase their stability in the circulation (Sanderson et al, 2005; Kellogg et al, 2011).…”

“…Normally, we consider the internalization that influences the efficacy of ADCs to be regulated by antigen. Recently, Lee et al (2018a) demonstrated that the internalization may be mainly determined by the cellular environment rather than the antigen, which brought another hint that the development of the ADCs has to consider a variety of parameters besides the choice of target and the design of the linker. The characteristics of tumor cells also affect the activity of ADCs, including the endothelium, interstitial, and epithelial barriers which could limit ADCs uptake in the tumor, resulting in a small fraction of the injected dose reaching the desired tumor target (Perez et al, 2014).…”

Improving Receptor-Mediated Intracellular Access and Accumulation of Antibody Therapeutics—The Tale of HER2

“…This approach removes necessary washing and cell-stripping steps used by studies in this review. If possible, one could consider the strategy by Lee et al, which incorporated fluorescence resonance energy transfer dyes into the linker connecting the antibody to the drug [136]. Using this approach, one can track both the antibody and payload inside cells.…”